Use of immunoblotting in testingMadurella mycetomatisspecific antigen

Abstract

Background: Though serodiagnosis of actinomycetoma is established, that of eumycetoma due to Madurella mycetomatis is limited because of lack of pure antigen. Reliable rapid tests are needed to make an accurate timely diagnosis. The purpose of this study is to detect antigen parts of M. mycetomatis, which act specifically with M. mycetomatis antibodies.Methods: Cytoplasmic antigen was prepared from molecularly identified cultures of M. mycetomatis by sonication, ultracentrifugation, dried, weighed and appropriately reconstituted. M. mycetomatis cytoplasmic antigen were separated using 12% sodium dodecyl sulfate-polyacrylamide gel, and immunoblotting to detect the reactive ones.Immunoblotting was carried out in nitrocellulose strips containing different molecular size. Sera from patients and co-patients as control were used.Results: When stained with Coomassie brilliant blue R 250 seven molecular weights appeared but only three, 45, 60, 95 kDa reacted with M. mycetomatis patients few from control group, one from a malaria patient. No reactive band was observed with sera from actinomycetoma, Aspergillus flavus-associated aspergillosis, schistosomiasis, leishmaniasis, fungal sinusitis nor healthy controls.Conclusions: Specific fractions of M. mycetomatis antigen which were demonstrated by immunoblotting showed 75% sensitivity and 95% specificity. The true negative tests were 14 patients (32.5%). This also means that immunoblotting is reasonably reliable in diagnosis and follow-up of eumycetoma patients.

The Diagnosis of Fungal Neglected Tropical Diseases (Fungal NTDs) and the Role of Investigation and Laboratory Tests: An Expert Consensus Report

“…But adapting or developing new diagnostic tests, drawing on the results of recent research was identified as a critical need for the future. For mycetoma, important recent developments with direct impact on diagnosis include molecular methods to identify new etiologic agents, isothermal amplification techniques, the adaptation of MALDI Tof to fungal identification and the finding of novel infection-related antigens [14][15][16][17]. A new grading system for Magnetic Resonance Imaging (MRI) allows clinicians to assess extent and severity of mycetoma, which may not be apparent on visual inspection [18].…”

Mycetoma laboratory diagnosis: Review article

“…ELbadawi and associates in 2016 reported on the 75% sensitivity and 95% specificity of immunoblotting of M . mycetomatis cytoplasmic antigen from molecularly identified cultures [ 29 ].…”

Diagnostics to support mycetoma management—Development of two target product profiles

“…For N. brasiliensis , diagnostic antigens have been identified that can be used in an enzyme‐linked immunosorbent assay (ELISA) [ 17 ] or a lateral flow device in the future. For M. mycetomatis , antigens have been identified, but these were not able to differentiate patients from healthy controls [ 18 ] or were not further characterised [ 19 ]. No antigens are available for the other causative agents, and antigen‐specific antibodies cannot be quantified in sera from patients with lateral flow assays or ELISAs.…”